PROJECT SUMMARY/ABSTRACT Normal hearing depends on the delicate mechanotransduction apparatus of inner ear hair cells ? the stereociliary hair bundle. Cochlear vibrations deflect the hair bundle, which converts mechanical stimulation to electrical signals via gating of mechanosensitive ion channels. Hair bundle transduction is critical for hearing, as it is required for afferent signaling by inner hair cells, and for activation of the electromotile response of the outer hair cells, which serve to amplify cochlear vibrations. However, it remains unclear whether the hair bundle simply detects the forces imposed on it by the surrounding cochlear structures, or if the mechanical properties of the hair bundle itself play an important role in shaping these vibrations. To address this, the proposed work will use an innovative, optical coherence tomography-based technique to measure sound- evoked vibrations in the intact cochleae of mice, including those with mutations targeting the hair bundle. Specifically, we will test the hypothesis that the passive, nonlinear stiffness of the hair bundle significantly influences cochlear vibrations. In Aim 1, we will estimate the passive hair bundle stiffness in wild-type CBA/CaJ mice, as well as mice lacking stereociliary tip links (Salsa mice) and rootlets (Triobp mice), so as to determine how these individual hair bundle components contribute to the total stiffness. Hair bundle stiffness will be calculated by dividing the force applied to the bundle, as estimated from a cochlear model, by the resulting hair bundle deflection, which will be estimated from measurements of the radial motions of the tectorial membrane and reticular lamina. In Aim 2, we will examine how passive hair bundle stiffness contributes to nonlinear distortion within the cochlea. Harmonic distortions will be examined in the vibrations of the basilar membrane, tectorial membrane, and reticular lamina in CBA/CaJ, Salsa, and Triobp mice. To examine the influence of the hair bundle alone, measurements will be made using conditions which minimize the influence of outer hair cell somatic electromotility, as well as in Prestin 499 knockin mice, which lack electromotility. Pursuit of these aims will reveal how specific hair bundle properties influence the magnitude and nonlinearity of vibrations within the cochlea, and will provide the first in vivo estimates of hair bundle stiffness, as well as the first detailed examinations of mechanical distortion in the mouse cochlea. The findings will be significant, as much of our knowledge about hair bundle mechanics comes from in vitro preparations where the hair bundle?s environment may differ considerably from that encountered in vivo.